Universal HRP-AEC Substrate Colorimetric Kit Product Manual (Chinese version)
The main purpose
The universal HRP-AEC substrate chromogenic reagent is a method designed to catalyze the peroxidase activity by oxidizing a substrate, 3-amino-9-ethylcarbazole, to form a red insoluble product. In turn, an authoritative and classical technical method for the expression of target molecules is determined. Its immunological assays for horseradish peroxidase-labeled proteins include protein Western hybridization and immunochemistry, as well as in situ hybridization staining. The product is ready to use, stable performance, convenient operation, high sensitivity, clear color and good repeatability.
technical background
3-amino-9-ethylcarbazole (AEC) is a chromogenic substrate for peroxidase having a molecular formula of C14H14N2 and a molecular weight of 210.3. Under the catalysis of peroxidase, the electron donor AEC substrate loses electrons under hydrogen peroxide to exhibit color change and deposition, forming a stable red insoluble product, but dissolved in an organic solvent. It is used to detect the activity of peroxidase with high sensitivity and specificity.
product content
Staining solution (Reagent A) 5 ml
Diluent (Reagent B) 90 ml
Reaction solution (Reagent C) 5 ml
Product manual 1 copy
storage method
Store in a 4°C refrigerator; Reagent A, avoiding light; effective for June
User-supplied
Non-ionized water: used to clean samples
PBS buffer solution: used to clean samples
Hematoxylin counterstaining kit (80067.2): used for counterstaining after routine dyeing
15 ml conical centrifuge tube: container for dyeing working fluid
Flat shaker: for incubation and mixing reactions
2.2 ml centrifuge tube: container for dyeing working fluid
Optical microscope: for observation and analysis after dyeing
Experimental procedure
- Immunoblot staining
Before the start of the experiment, transfer 500 μl of staining solution (Reagent A) to a 15 ml conical centrifuge tube, add 9 ml of diluent (Reagent B), mix, add 500 μl of reaction solution (Reagent C) , and mix. After that, mark it as a dyeing solution to avoid light. Then do the following.
- Prepare a solid phase membrane (5 X 8 cm 2 ) containing the protein to be tested: including electrophoresis, transfer, blocking, primary antibody (or biotin) treatment, horseradish peroxidase (HRP) labeled secondary antibody (or Streptavidin) treatment, cleaning, etc.
- Add 5 ml of room temperature preheated dyeing working solution to cover the entire surface of the solid phase membrane
- Place on a flat shaker, incubate for 5 to 10 minutes at room temperature, or until red precipitate appears; avoid light
- Clamp the solid phase membrane with tweezers and put it in non-ionized water.
- Dry in the air
- Digital camera photo recording
- Immunochemical staining
Before the start of the experiment, transfer 100 μl of the staining solution (Reagent A) to a 2.2 ml centrifuge tube, add 1.8 ml of the diluent (Reagent B) , mix, add 100 μl of the reaction solution (Reagent C) , and mix well. Marked as a dyeing working solution and placed in a dark room. Then do the following:
- Prepare tissue sections containing the protein to be tested: including fixation, blocking, primary antibody (or biotin) treatment, horseradish peroxidase (HRP) labeled secondary antibody (or streptavidin) treatment, washing, etc.
- At room temperature, carefully add 100 μl of dyeing solution to cover the entire surface of the sample.
- Incubate for 5 to 30 minutes at room temperature or until the sample appears red
- Carefully remove the staining solution from the slice
- At room temperature, carefully place the slices in 50 ml of user-prepared PBS buffer for 5 seconds.
- Carefully remove the PBS buffer solution from the section
- (Selection step) for hematoxylin counterstaining (Note: Hematoxylin counterstaining kit -80050 is recommended )
- Put a cover slip or cover
- Immediately observed under a general optical microscope: positive red
Precautions
- This product is 10 times (10 ml / time) or 1000 times (100 microliters / time) operation
- Can be used on 1000 cm 2 immunoblotting membranes: 5 ml is recommended for a 5 x 8 cm 2 patch
- Can be used for 1000 pieces of 1 cm X 1 cm tissue sections
- Users can adjust the reagent dosage according to their needs.
- Wear gloves when handling
- The dyeing time should be adjusted according to the situation, so it should not be too long to avoid excessive color development.
- If the closure is not good, it will easily cause the background color to be too dark.
- The dyeing working solution is freshly prepared and should not be more than 1 hour; if precipitation is found, it should not be used.
- The solid phase film will fade after several hours of dyeing and cannot be permanently present.
- This product can only be used for hybridization and precipitation reactions of horseradish peroxidase (HRP) labeling.
- After dyeing, it can be counterstained with hematoxylin to enhance contrast; it is recommended to use MAYER'S hematoxylin counterstaining kit-80067.2
- Cannot be cleaned with organic solvents such as ethanol after dyeing
- The company provides a series of dyeing reagents
Quality Standard
- This product has been certified to be stable.
- This product has been identified and clearly colored
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